Abstract：Objective To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology. Methods At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice. Results The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P < 0.001). Conclusions A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.
MA Fei,SHI Cong-Cong,LIANG Pu-Ping et al. Construction of a mouse model of cblC type methylmalonic acidemia with W203X mutation based on the CRISPR/Cas9 technology[J]. CJCP, 2019, 21(8): 824-829.
Fowler B, Leonard JV, Baumgartner MR. Causes of and diagnostic approach to methylmalonic acidurias[J]. J Inherit Metab Dis, 2008, 31(3):350-360.
[5]
Lerner-Ellis JP, Tirone JC, Pawelek PD, et al. Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type[J]. Nat Genet, 2006, 38(1):93-100.
Liu MY, Yang YL, Chang YC, et al. Mutation spectrum of MMACHC in Chinese patients with combined methylmalonic aciduria and homocystinuria[J]. J Hum Genet, 2010, 55(9):621-626.
Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121):819-823.
[10]
Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096):816-821.
[11]
Zhou X, Cui Y, Han J. Methylmalonic acidemia:current status and research priorities[J]. Intractable Rare Dis Res, 2018, 7(2):73-78.
[12]
Fischer S, Huemer M, Baumgartner M, et al. Clinical presentation and outcome in a series of 88 patients with the cblC defect[J]. J Inherit Metab Dis, 2014, 37(5):831-840.
[13]
Nogueira C, Marcão A, Rocha H, et al. Molecular picture of cobalamin C/D defects before and after newborn screening era[J]. J Med Screen, 2017, 24(1):6-11.
[14]
Forny P, Schumann A, Mustedanagic M, et al. Novel mouse models of methylmalonic aciduria recapitulate phenotypic traits with a genetic dosage effect[J]. J Biol Chem, 2016, 291(39):20563-20573.
[15]
Peters HL, Pitt JJ, Wood LR, et al. Mouse models for methylmalonic aciduria[J]. PLoS One, 2012, 7(7):e40609.
[16]
Peters H, Nefedov M, Sarsero J, et al. A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality[J]. J Biol Chem, 2003, 278(52):52909-52913.
[17]
Yang Y, Wang L, Bell P, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice[J]. Nat Biotechnol, 2016, 34(3):334-338.
[18]
Singh K, Evens H, Nair N, et al. Efficient in vivo liver-directed gene editing using CRISPR/Cas9[J]. Mol Ther, 2018, 26(5):1241-1254.
[19]
An D, Schneller JL, Frassetto A, et al. Systemic messenger RNA therapy as a treatment for methylmalonic acidemia[J]. Cell Rep, 2017, 21(12):3548-3558.
